We report that overexpression of Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3), here identified in a 38-y-old woman suffering from long-standing fatigue and exercise intolerance, can disrupt mitochondrial respiratory supercomplex formation and is associated with endoplasmic reticulum (ER) stress. Increased expression of WASF3 in transgenic mice markedly decreased their treadmill running capacity with concomitantly impaired respiratory supercomplex assembly and reduced complex IV levels in skeletal muscle mitochondria. WASF3 induction by ER stress using endotoxin, well known to be associated with fatigue in humans, also decreased skeletal muscle complex IV levels in mice, while decreasing WASF3 levels by pharmacologic inhibition of ER stress improved mitochondrial function in the cells of the patient with chronic fatigue.